The invention relates to a starch conversion process of the type which includes a debranching step. The invention also relates to the use of a thermostable isoamylase for debranching starch. The invention further relates to an isolated isoamylase obtained from a strain of the genus Rhodothermus, to cloned DNA sequences encoding isoamylases derived from a strain of Rhodothermus or Sulfolobus, to expression vectors comprising said DNA sequence, host cells comprising such expression vectors, and finally to methods for producing said isoamylases.
Starches such as corn, potato, wheat, manioc and rice starch are used as the starting material in commercial large scale production of sugars, such as high fructose syrup, high maltose syrup, maltodextrins amylose, trehalose, G2-G8 oligosaccharides (including functional oligosaccharides) and other carbohydrate products such as fat replacers.
Degradation of Starch
Starch usually consists of about 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains xcex1-1,4 D-glucose residues are joined by xcex1-1,6 glucosidic linkages. Amylopectin is partially degraded by xcex1-amylase which hydrolyze the 1,4-xcex1-glucosidic linkages into branched and linear oligosaccharides. Prolonged degradation of amylopectin by xcex1-amylase results in the formation of so-called xcex1-limit dextrins which are not susceptible to further hydrolysis by the xcex1-amylase. Branched oligosaccharides can be hydrolyzed into linear oligosaccharides by a debranching enzyme. The remaining branched oligosaccharides can slowly be depolymerized to D-glucose by glucoamylase. Glucoamylase hydrolyse linear oligosaccharides fast into D-glucose.
Amylose is a linear polysaccharide built up of D-glucopyranose units linked together by xcex1-1,4 glucosidic linkages. Amylose is degraded into linear oligosaccharides by xcex1-amylase which are fast depolymerized into D-glucose by glucoamylase.
Enzymes
xcex1-amylase
xcex1-amylase (E.C. 3.2.1.1) with the systematically name 1,4-xcex1-D-glucan glucanohydrolase is capable of hydrolysing starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. xcex1-amylase acts on the substrate in a random manner. The reducing groups are liberated in the xcex1-configuration.
Debranching Enzymes
Debranching enzymes, which can attack amylopectin are divided into two classes: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41), respectively. Isoamylase hydrolyses xcex1-1,6-D-glucosidic branch linkages in amylopectin and xcex2-limit dextrins and can be distinguished from pullulanases by the in-ability of isoamylase to attack pullulan, and by the limited action on xcex1-limit dextrins.
Glucoamylase
Glucoamylase or glucan 1,4-xcex1-glucosidase (E.C. 3.2.1.3) hydrolyzes the terminal 1,4-linked xcex1-D-glucose residues successively from the non-reducing ends of the chains with release of xcex2-D-glucose. The enzyme can also slowly hydrolyze 1,6-xcex1-D-glucosidic bonds in isomaltose, panose and related oligosaccharides.
Starch Conversion
A xe2x80x9ctraditionalxe2x80x9d starch conversion process degrading starch to lower molecular weight carbohydrate components such as sugars or fat replacers includes a debranching step.
Starch to Sugar Conversion
In the case of converting starch into a sugar the starch is depolymerized. A such depolymerization process consists of a pretreatment step and two or three consecutive process steps, viz. a liquefaction process, a saccharification process and dependent on the desired end product optionally an isomerization process.
Pre-treatment of Native Starch
Native starch consists of microscopic granules which are insoluble in water at room temperature. When an aqueous starch slurry is heated, the granules swell and eventually burst, dispersing the starch molecules into the solution. During this xe2x80x9cgelatinizationxe2x80x9d process there is a dramatic increase in viscosity. As the solids level is 30-40% in a typically industrial process, the starch has to be thinned or xe2x80x9cliquefiedxe2x80x9d so that it can be handled. This reduction in viscosity is today mostly obtained by enzymatic degradation.
Liquefaction
During the liquefaction step, the long chained starch is degraded into branched and linear shorter units (maltodextrins) by an xcex1-amylase (e.g. Termamyl(trademark)). The liquefaction process is carried out at 105-110xc2x0 C. for 5 to 10 minutes followed by 1-2 hours at 95xc2x0 C. The pH lies between 5.5 and 6.2. In order to ensure an optimal enzyme stability under these conditions, 1 mM of calcium is added (40 ppm free calcium ions). After this treatment the liquefied starch will have a xe2x80x9cdextrose equivalentxe2x80x9d (DE) of 10-15.
Saccharification
After the liquefaction process the maltodextrins are converted into dextrose by addition of a glucoamylase (e.g. AMG(trademark)) and a debranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or a pullulanase (e.g. Promozyme(trademark)) (U.S. Pat. No. 4,560,651). Before this step the pH is reduced to a value below 4.5, maintaining the high temperature (above 95xc2x0 C.) to inactivate the liquefying xcex1-amylase to reduce the formation of short oligosaccharide called xe2x80x9cpanose precursorsxe2x80x9d which cannot be hydrolyzed properly by the debranching enzyme.
The temperature is lowered to 60xc2x0 C., and glucoamylase and debranching enzyme are added. The saccharification process proceeds for 24-72 hours.
Normally, when denaturing the xcex1-amylase after the liquefaction step about 0.2-0.5% of the saccharification product is the branched trisaccharide 62-xcex1-glucosyl maltose (panose) which cannot be degraded by a pullulanase. If active amylase from the liquefaction step is present during saccharification (i.e. no denaturing), this level can be as high as 1-2%, which is highly undesirable as it lowers the saccharification yield significantly.
Isomerization
When the desired final sugar product is e.g. high fructose syrup the dextrose syrup may be converted into fructose. After the saccharification process the pH is increased to a value in the range of 6-8, preferably pH 7.5, and the calcium is removed by ion exchange. The dextrose syrup is then converted into high fructose syrup using, e.g., an immmobilized glucoseisomerase (such as Sweetzyme(trademark)).
Starch to Fat Replacer Conversion
A xe2x80x9cfat replacerxe2x80x9d is a fat-like carbohydrate which is used as a functional replacement of fat in foods. Fat-replacers typically consist of short chained amylose of linear polymers containing from about 15 to 65 anhydroglucose units linked by xcex1-1,4-D-glucosidic bonds. Such fat replacers may be produced by enzymatic debranching of starch. Methods for degrading the xcex1-1,6-D-glucosidic bonds of starch to form short chain low molecular weight amylose by the use of debranching enzymes are described in e.g. U.S. Pat. No. 3,730,840 (Sugimoto), U.S. Pat. No. 3,881,991 (Kurimoto) and U.S. Pat. No. 3,879,212 (Yoshida). A further method of producing short chained amylose to be used as a fat replacer is described in EP 0,486,936-A1 (National Strach and Chemical Investment Holding Corporation).
It is the object of the present invention to provide a starch conversion process of the type which includes a debranching step which results in a reduced formation of the undesired trisaccharide panose. The invention also relates to a novel thermostable isoamylase suitable for use in the starch conversion process of the invention.
According to the invention the term xe2x80x9cstarch conversion processxe2x80x9d include all processes where starch is degraded into carbohydrate components with a lower molecular weight.
The present inventors have found that the achievement of the above-mentioned object of the invention requires an isoamylase debranching enzyme which is active at the process condition prevailing.
In the first aspect the invention relates to a starch conversion process of the type which includes a debranching step wherein an isoamylase being active at the process conditions prevailing is used for debranching the starch.
In the second aspect the invention relates to the use of a thermostable isoamylase in starch conversion processes.